3 four lane flow-cell chips
6 lane stickers
2 stage securing magnets
EV Profiler Kit V2
Quantify your EVs, Inside and Out
Harness the power of single-molecule localization microscopy to characterize EVs across scales. Quantify nanometer small, rare, and precious EV samples with super-resolution precision. ONI’s EV Profiler helps you focus on answering the right questions about your EV populations.
What can you do with the EV Profiler Kit V2?
Confidently identify and size EVs
We understand how important it is for EV researchers to assess the quality of their EV samples, understand EV heterogeneity and determine EV morphology. The current MISEV guidelines outline that the combination of protein and non-protein markers when colocalized can provide greater evidence of EV specificity in labeling for single particle measurements. This is why EV Profiler V2 includes an ONI developed pan-EV stain, a lectin-based dye that binds glycoproteins of EV membranes. Together with validated antibodies against common tetraspanin markers (CD9, CD63 and CD81), the kit provides the tools to confidently identify EVs and determine the size distribution of EV populations.
An ONI developed pan-EV stain binds EV membrane glycoproteins. Together with validated antibodies against tetraspanin markers this allows confident EV identification and more accurate assessment of EV morphology sizing. Image above shows HCT116 cell line-derived EVs labeled with pan-EV stain (yellow), anti-CD63 (cyan) and anti-CD81 (magenta). Graphs show two different EV populations sized based on tetraspanin (left) or pan-EV signal (right).
Detection of EV cargo and surface biomarkers on the same sample
The EV Profiler Kit V2 is able to detect and quantify luminal protein cargo on single EVs, as well as surface biomarkers. Up to 3 markers can be detected within the same sample.
The EV Profiler Kit V2 is able to detect and quantify luminal protein cargo on single EVs, as well as surface biomarkers. HCT116-derived EVs were stained with ONI’s pan-EV stain labeled with 488, Tetraspanin Trio (anti-CD9, anti-CD63 and anti-CD81 all conjugated to 568 and anti-Syntenin-1 labeled with 647. Graphs show the number of Tetraspanins or Syntenin-1 counts from EVs positive for either or both markers. Permeabilization increases the overlap of Tetraspanin and Syntenin-1 counts from 0.61% to 35.8% (percentages not shown).
Assess EV morphology
Single molecule localization microscopy used in combination with the appropriate fluorescently labeled probes, helps assess the integrity and morphology of EV populations and single EVs after purification. ONI’s pan-EV stain in combination with cluster-based analysis on our CODI cloud platform, allows reporting of morphological features of the EV, including size and circularity.
The EV Profiler Kit V2 is powered by ONI’s CODI cloud software, which enables reporting of EV morphological features based on clustering of single molecule data. The different EV morphologies within heterogeneous EV populations can be determined with features such as diameter and circularity; two examples are shown with images superimposed to the graph with A representing a smaller and less circular EV, and B representing a larger more circular EV.
Characterize across-scales: from EV populations down to single EVs
Study EV populations across scales. Each EV Profiler kit contains enough material to analyze 12 EV samples using our 4 lane analysis chip which has been developed and optimized for EV capture. You can capture and image hundreds of EVs from any given source with the appropriate capture method (read more details below), then zoom into individual EVs to see biomarker distribution at a single-EV level.
ONI’s Nanoimager and CODI platform enable imaging and visual inspection of EV samples across scales. Scale bars range from 10 µm to 50 nm when inspecting one EV out of thousands of EVs imaged on from an HCT116-derived EV sample captured with ONI’s S4 molecule and labeled with pan-EV stain (yellow), anti-CD63-568 (cyan) and anti-CD81-647 (magenta).
Obtain rapid results with a benchtop assay
Capture, stain, image and quantify
EV Profiler V2 contains all necessary reagents to capture, fix and stain EVs ready for imaging in a same-day workflow for single EV characterization. The workflow works with EV sample volumes as low as 10 µL.
Single molecule localization data and nanoscale resolution images can be obtained using the Nanoimager, our benchtop super-resolution microscope. Data is then analyzed through our CODI cloud-based software, making it possible to get results within a few hours from start to finish.
Flexibility to suit your EV samples
Multiple capture strategies
EV Profiler offers 3 options for EV capture on the passivated glass surface of the assay chips:
Tetraspanin based capture
ONI validated capture antibodies against tetraspanins: anti-CD9, anti-CD63 and anti-CD81 antibodies are included in the kit
Phosphatidylserine-dependent, protein-independent capture
Our proprietary S4 capture molecule binds EVs in a protein-independent manner, working best with phosphatidylserine-rich EVs.
Custom target based capture
Use your own antibody of choice against your target of interest – it simply needs to be biotinylated (sample above provided by Dr Nicole Meisner-Kober’s lab).
Swap our antibodies for your own easily
In the EV Profiler Kit V2 antibodies for standard tetraspanin markers (CD9, CD63, and CD81) are included conjugated to the same fluorophore to be used as Tetraspanin Trio. This enables researchers to use two channels for their own biomarkers of interest if needed. We have built a modular kit where antibodies can be swapped for your own validated antibodies against targets of interest inside or outside EVs. These need to be conjugated to dSTORM-compatible dyes.
You can find more information on fluorophores for dSTORM imaging here. The kit’s sample preparation protocol will guide you through the steps for incorporating your antibodies into the experiment and our image acquisition and analysis guide will provide you with the information needed when optimizing your antibodies.
| Configuration example | 488 channel | 568 channel | 647 channel |
|---|---|---|---|
| Kit components + antibody of choice | Pan-EV stain-488 | Tetraspanin Trio 568 | own antibody 647 |
| Kit components + cargo Add-on | Pan-EV stain-488 | Tetraspanin Trio 568 | anti-Syntenin-1 647 |
| Other tetraspanin Add-ons | anti-CD9 488 | anti-CD63 568 | anti-CD81 647 |
| Two antibodies of choice | own antibody 488 | Tetraspanin Trio 568 | own antibody 647 |
| Two antibodies of choice | Pan-EV stain-488 | own antibody 568 | own antibody 647 |
EV analysis and quantification made simple
Purchase of the kit includes access to purpose built analysis tools within our CODI cloud platform, which allow seamless processing of single molecule localization data to obtain output summary data files.
- Seamless upload data of acquired data to CODI cloud platform
- Visualization of individual EVs and their biomarkers with the nanoscale resolution
- Analysis tools tailored to EV workflow:
- Correction of spatial drift
- Filtering of individual localizations
- Cluster-based single molecule
- Extract quantitative data on EV populations based on raw data outputs (.csv format):
- Biomarker positivity per sample
- EV size distribution
- Biomarker density per EV (localizations/cluster)
- EV morphology parameters
- Export high-resolution 3-color images from your EV samples
ONI’s CODI cloud software uses clustering of single molecule data to enable study of EVs. Left image shows EVs captured using the EV Profiler Kit V2, which were imaged with the Nanoimager. Single molecule localization data is then clustered on CODI to help assess EV morphology features and determine levels of positivity for different biomarkers.
What our customers are saying!
EV Profiler is powered by ONI’s Nanoimager and CODI platform
Combine the advantages of super-resolution imaging with powerful analysis of larger numbers of events. This allows detection and quantification of biomarker abundance with up to 3 biomarkers detected within the same sample using dSTORM, and reporting of EV morphology, including size and circularity.
Whats in the box?
Chips |
|
| 3 four lane flow-cell chips | |
| 6 lane stickers | |
| 2 stage securing magnets | |
Capture reagents |
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| S4 capture (3 tubes) | |
| Biotin capture CD9 (1 tube) | |
| Biotin capture CD63 (1 tube) | |
| Biotin capture CD81 (1 tube) | |
Detection probes |
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| Pan-EV stain 488 (3 tubes) | |
| Tetraspanin trio 568 (3 tubes, 3 antibodies mixed) | |
Standards |
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| E1 standard EVs (1 tube) | |
Buffers |
|
| S3, W1, F1, N1, P1, W2, C1 (3 tubes each) | |
| BCubed imaging buffer parts A and B (3 tubes each) | |
Add-ons |
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| Anti-Syntenin-1 647 | |
| Anti-CD9 568 | |
| Anti-CD63 568 | |
| Anti-CD81 568 | |
| Anti-CD9 488 | |
| Anti-CD81 647 | |
| Biotin anti-CD9 | |
| Biotin anti-CD63 | |
| Biotin anti-CD81 | |
S4 capture (3 tubes)
Biotin capture CD9 (1 tube)
Biotin capture CD63 (1 tube)
Biotin capture CD81 (1 tube)
Pan-EV stain 488 (3 tubes)
Tetraspanin trio 568 (3 tubes, 3 antibodies mixed)
E1 standard EVs (1 tube)
S3, W1, F1, N1, P1, W2, C1 (3 tubes each)
BCubed imaging buffer parts A and B (3 tubes each)
